Introduction and Spread of Dengue Virus 3, Florida, USA, May 2022–April 2023

During May 2022–April 2023, dengue virus serotype 3 was identified among 601 travel-associated and 61 locally acquired dengue cases in Florida, USA. All 203 sequenced genomes belonged to the same genotype III lineage and revealed potential transmission chains in which most locally acquired cases occurred shortly after introduction, with little sustained transmission.


Dengue virus sequencing
At the Yale School of Public Health, dengue virus RNA was sequenced using a panserotype, highly multiplexed PCR approach, called 'DengueSeq' (a derivative of PrimalSeq) (1).
A detailed protocol, including the DENV1-4 primer schemes, was previously published (2).In brief, we prepared libraries using the Illuminia COVIDSeq test (RUO version) with the DENV1-4 primers, and pooled libraries were sequenced on the Illumina NovaSeq (paired-end 150), targeting 1 million reads per individual library.Consensus genomes were generated at a depth of coverage of 10X and minimum frequency threshold of 0.75 using a custom DengueSeq bioinformatics pipeline (3), which includes iVar (4).All DENV genomes and sequencing data are available on BioProject PRJNA951702.
At the CDC Dengue Branch, dengue virus RNA was sequenced using dengue virus serotype 3 (DENV-3) specific custom-designed PCR primers.A detailed protocol was previously described (5).Briefly, we prepared libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina with DENV-3 primers, and pooled libraries were sequenced on the Illumina MiSeq (paired-end 600).Consensus genomes were generated using a custom DengueSeq bioinformatics pipeline, which includes iVar.For the distributions of S and G, we applied a pruning approach to correct for biologically unlikely links by setting cutoff values.For the estimated serial interval, we removed all potential case connections with onset dates less than five days apart, and for both the temporal and genetic distance distributions under each assumed reporting probability we removed all pairs greater than the upper 95% quantile (Figure S3).The expected daily mutation rate was 1.9 × 10 -2 substitutions per day given the mean number of expected substitutions per nucleotide per year (6.98x10 -4 [95% PI: 5.39 x10 -4 -8.62x10For travel-associated cases, we did not attempt to identify the source of their infection as we assumed they were infected outside of Florida.Most compatible linkages for locally acquired cases were identified by identifying the linkage with the highest compatibility score.

Ethics
Sequencing of de-identified remnant clinical specimens that tested positive for dengue virus was done at the Yale School of Public Health.The Institutional Review Boards (IRB) from the Yale University Human Research Protection Program determined that pathogen genomic sequencing of de-identified remnant diagnostic samples as conducted in this study is not research involving human subjects (Yale IRB Protocol ID: 2000033281).

Transmission linkage compatibility score calculation
Using sequenced locally acquired and travel-associated cases reported with an onset date in 2022, we reconstructed possible transmission chains building upon methods published inHampson et al., 2009 (6)  andCori et al., 2018 (7).For each reported case (i), we calculated a compatibility score (pij) for linkage between the case and its potential sources (j) defined as all cases with an onset date preceding case i as follows:S and G represent the probability density function of the dengue serial interval (Gamma distributed) and probability mass function of the case genetic distance accounting for the serial interval (Negative binomial distributed), respectively.Here tij and dij represent the temporal and genetic distance between cases i and j, whilst x indicates all possible values of each probability function.To account for unobserved transmission between i and j, we expand the default probability functions by the reporting probability as described in Cori et al.,2018.Geographic location was not accounted for when determining the compatibility score.
-4 ]) and the whole genome size (10,170) based off the molecular analysis done earlier in this work.To infer parameter values for the serial interval, we fitted a Gamma distribution to 1,000 simulated sums of extrinsic and intrinsic incubation periods of dengue virus (Chan & Johansson, 2012) (8) and a mosquito half-life assuming temperatures of 20-30 degrees Celsius(Brady et al., 2013) (9).